Field of the Invention
This invention relates to the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated candidate strain vaccine for the highly virulent Georgia 2007 isolate ASFV-G. The vaccine comprises the ASFV-G ΔMGF, a recombinant ASFV-G modified by deleting a large portion of the MGF (Multi Gene Family) genes.
Description of the Relevant Art
African Swine Fever (ASF) is a contagious viral disease of swine. The causative agent, ASF virus (ASFV), is a large enveloped virus containing a double-stranded DNA genome of approximately 190 kilobase pairs. ASFV shares aspects of genome structure and replication strategy with other large double-stranded DNA viruses, including the Poxviridae, Iridoviridae and Phycodnaviridae (Costard et al. 2009. Phil. Trans. Royal Soc. B 364:2683-2696). ASFV infections in domestic pigs are often fatal and are characterized by fever, hemorrhages, ataxia and severe depression. However, the course of infection varies, ranging from highly lethal to sub-clinical, depending on host characteristics and the particular virus strain (Tulman et al. 2009. Curr. Top. Microbiol. Immunol. 328:43-87).
Currently, the disease is endemic in more than twenty sub-Saharan African countries. In Europe, ASF is still endemic on the island of Sardinia (Italy) and new outbreaks have been declared in the Caucasus region since 2007, affecting Georgia, Armenia, Azerbaijan and Russia. Isolated outbreaks have been recently reported in Ukraine, Belarus, Lithuania, Latvia and Poland, posing the risk of further dissemination into neighbouring countries. The epidemic virus, ASFV Georgia 2007/1, is a highly virulent isolate belonging to the genotype II (Chapman et al. 2011. Emerging Infect. Dis. 17:599-605).
At present, there is no vaccine available for ASF and disease outbreaks are controlled by animal quarantine and slaughter. Attempts to vaccinate animals using infected cell extracts, supernatants of infected pig peripheral blood leukocytes, purified and inactivated virions, infected glutaraldehyde-fixed macrophages, or detergent-treated infected alveolar macrophages failed to induce protective immunity (Coggins, L. 1974. Prog. Med. Virol. 18:48-63; Forman et al. 1982. Arch. Virol. 74:91-100; Kihm et al. 1987. In: African Swine Fever, Becker, Y. (ed), Martinus Nijhoff, Boston, pp 127-144; Mebus, C. A. 1988. Adv. Virus Res. 35:251-269). Homologous protective immunity does develop in pigs surviving viral infection. Pigs surviving acute infection with moderately virulent or attenuated variants of ASFV develop long-term resistance to homologous, but rarely to heterologous, virus challenge (Hamdy and Dardiri. 1984. Am. J. Vet. Res. 45:711-714; Ruiz-Gonzalvo et al. 1981. In: FAO/CEC Expert Consultation in ASF Research, Wilkinson, P. J. (ed), Rome, pp 206-216). Pigs immunized with live attenuated ASF viruses containing engineered deletions of specific ASFV virulence-associated genes were protected when challenged with homologous parental virus. Specifically, individual deletion of UK (DP69R), 23-NL (DP71L), TK (A240L) or 9GL (B119L) genes from the genomes of pathogenic ASF viruses (Malawi Lil-20/1, Pretoriuskop/96/4, and E70) markedly attenuated the virus in swine and the animals immunized with these attenuated viruses were protected against challenge with homologous virus (Moore et al. 1998. J. Virol. 72:10310-10315; Lewis et al. 2000. J. Virol. 74:1275-1285; Zsak et al. 1996. J. Virol. 70:8865-8871; Zsak et al. 1998. J. Virol. 72:1028-1035). These observations constitute the only experimental evidence describing the rational development of an effective live attenuated virus against ASFV.
Variations in genome size and restriction fragment patterns are observed among different ASFV isolates and this diversity resides in the terminal genomic regions. These ASFV variable regions comprise the left 35-kb and the right 15-kb ends of the genome and contain at least five multigene families (MGFs): MGF 100, MGF 110, MGF 300, MGF 360, and MGF 505 (Neilan et al. 2002. J. Virology 76:3095-3104). These genes are grouped in these MGFs because the share sequence and structural identity. The functions of these genes are not completely understood, but they have been correlated with macrophage host range, modulation of the innate host immune response and virulence (Zsak et al. 2001. J. Virol. 75:3066-3076; Afonso et al. 2004. J. Virol. 78:1858-1864; Neilan et al. 2002, supra).
Deletions of several MGF genes in association with the deletion of the NL gene (resulting in the deletion of a total of 7,559 nucleotides) have been used to attenuate the virulent isolate Malawi already having deleted its NL gene (Neilan et al. 2002, supra). However, the resulting attenuated Malawi-ΔNL-ΔMGF ASFV was not tested as an experimental vaccine to assess its ability to prevent disease when challenged with the virulent parental virus. Nor was the effect of the modified virus against heterologous ASFV strains determined. Thus, there is a need for an effective live attenuated vaccine for the highly virulent ASFV Georgia 2007 isolate, ASFV-G, for which there is no vaccine candidate.